DNA, RNA, immunoglobulins, and proteins are classes of polymeric biomolecules (“biopolymers”) of particular importance in modern biochemical and molecular biological methods and processes. Specifically, biopolymers play critical roles in various subcellular processes including the preservation and transmission of genetic information, the production of proteins, and the formation of enzymes.
Due to the importance of these biopolymers in various biological processes, a wide variety of techniques have been developed to physically bind these classes of molecules in order to manipulate them for immobilization, purification, and concentration, etc. For example, various column methods have been developed to bind a biopolymer to a matrix with affinity for that biopolymer, thereby allowing for its immobilization, its separation from contaminating cellular components, its concentration, etc. See, for example, U.S. Pat. No. 5,652,141 for the purification of nucleic acids. Similarly, various bulk separation methods have been developed for the immobilization, separation, or concentration of biopolymers. For example, U.S. Pat. No. 6,718,742 describes the use of magnetic beads comprising a magnetic or paramagnetic particle and an attached ion exchange material capable of binding the biomolecule.
Biopolymer immobilization, separation, concentration or purification is employed across a wide range of commercial applications, including, for example, forensics, pharmaceutical research and development, medical diagnostics and therapeutics, environmental analysis, such as water purification or water quality monitoring, nucleic acid purification, proteomics, and field collection of biological samples. Thus, a need exists for efficient, simplified processing of clinical, environmental and forensic samples, especially for samples containing only nanogram amounts of nucleic acid or protein.
In contrast to conventional nucleic acid extraction and purification methodologies, sub-micron particles or nanoparticles as the solid phase platform for chromatography are provided. One of the preferred nanoparticle materials, kaolin, is used industrially as a filler and bonding agent and is commercially available at high purity and very low cost. The coupling process that has been developed provides surface modified particles (e.g., epoxy-silane coated kaolin) batch-wise in kilogram quantities under conditions where the production cost for the final product is dominated by quality control/quality assurance and packaging.
Various embodiments provide a unique three-phase chromatography based on highly purified, nearly mono-dispersed, ceramic or ceramic-like nanoparticles. These nanoparticles form stable colloidal suspensions in aqueous solution. Since the particles are sub-micron in diameter, they display a large surface area to volume ratio. For example, a milligram of kaolin nanoparticles of 200 nm diameter in colloidal suspension displays a total surface area in the range of 200 cm2, as surface area displayed by approximately 1012 dispersed nanoparticles. The inter-particle spacing between this number of particles suspended in a milliliter is about one micron, a distance that is less than the distance a 10,000 base pair long DNA molecule or a 1,000,000 Dalton protein would travel by passive diffusion in about one minute. Thus, even at a very low particle-mass density, and in the absence of mixing or convective flow, a 0.1% by weight suspension of these 200 nm diameter nanoparticles are at a per volume concentration such that a targeted biomolecule is never more than “a minute away” from colliding with the surface of a nanoparticle in colloidal suspension. This suggests that, independent of sample concentration, the binding phase for batch chromatography based on these colloidal suspended nanoparticles can be complete within minutes. In addition, due to the small size of these particles, the sedimented pellet volume of this 0.1% suspension of 200 nm kaolin nanoparticles may be as little as about one microliter. These ceramic nanoparticles, having an expansive surface area, provide very useful characteristics as the basis for chromatography, namely an enormous binding capacity per unit mass and the ability to be modified via well-known surface modifying chemistry.
For batch chromatography, the outer surface area is important because it defines the mass and the volume of sample that can be processed at one time. The surface area per unit mass increases with the inverse of the diameter (1/diameter). A comparison can be made between kaolin nanoparticles, having a surface area to mass ratio of about 200 cm per milligram, to smooth, non-porous, 30 micron diameter bead or glass particles, having approximately the same density as kaolin particles and a surface area to mass ratio of about 2 cm2 per milligram. About 100 milligrams of beads would be required to match the surface area of 1 milligram of the kaolin nanoparticles. Assuming that each type of matrix occupies about the same space per unit mass as a pellet, the surface area presented by 1 μL of a 200 nm nanoparticle pellet is equivalent to that of a 100 μL pellet for the standard 30 micron bead. Thus, in this example, a biologically relevant sample would have been concentrated 1000-fold via nanoparticles, but only 10-fold by the 30 μm beads. Alternatively, in terms of binding capacity, a 100 μL pellet of glass beads would have about the same total binding capacity as 1 μL of the nanoparticles.
Rapid development of a broad ranging nano-chromatography platform requires the ability to chemically modify the surface of the ceramic matrix with biomolecule-specific ligands. For these ceramic nanoparticles, surface chemistry has been studied and has been optimized for various applications in the plastics and polymers industry. The present invention utilizes ceramic surface chemistry and biochemical chromatography to develop a flexible repertoire of surface coatings for DNA, RNA, immunoglobulin, and protein applications, each based on the same underlying ceramic nanoparticle matrix.